14-3-3 mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels
14-3-3 mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels
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DOI：
10.1021/bi052640q
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发表时间：
2006-05-30
期刊：
影响因子：
2.9
通讯作者：
Ichimura, Tohru
中科院分区：
文献类型：
--
作者：
Nagaki, Kazunori;Yamamura, Hisao;Ichimura, Tohru
虽然最近的研究表明14-3-3蛋白是泛素E3蛋白连接酶的负调控因子，但其分子机制仍不清楚。我们先前证明14-3-3特异性结合E3酶之一Nedd 4 -2（KIAA 0439的人基因产物，称为hNedd 4 -2），其可以被血清糖皮质激素诱导蛋白激酶1（SGK 1）磷酸化;这种结合保护磷酸化/失活的hNedd 4 -2免于磷酸酶催化的去磷酸化[Ichimura，T.，等人（2005）J.Biol.Chem.280，13187-13194]。在这里，我们报告了14-3-3介导的hNedd 4 -2调节的另一种机制。使用表面等离子体共振光谱，我们表明14-3-3抑制hNedd 4 -2的WW结构域和上皮Na+通道ENaC的PY基序之间的相互作用。抑制作用呈剂量依赖性，并依赖于位于WW结构域之间的Ser 468的SGK 1催化磷酸化。重要的是，hNedd 4 -2的突变体，它可以被SGK 1磷酸化，但不能结合14-3-3，减少SGK 1介导的刺激非洲爪蟾卵母细胞中的ENaC诱导的电流。此外，14-3-3对已被cAMP依赖性蛋白激酶（PKA）磷酸化的hNedd 4 -2具有类似的作用。我们的结果，连同最近发现的14-3-3/parkin相互作用[Sato，S.，等人（2006）EMBO J. 25，211-221]，表明14-3-3通过抑制酶/底物复合物的形成来抑制泛素E3连接酶活性。
Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na+ channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.